Nobuko Obara

Apoptosis in mouse taste buds after denervation.

Abstract.Apoptotic cells in the taste buds of mouse circumvallate papillae after the sectioning of bilateral glossopharyngeal nerves were examined by the method of DNA nick-end labeling (TUNEL), together with standard electron microscopy. The taste buds decreased in number and size 3–11 days after denervation and disappeared at 11 days. The TUNEL method revealed only a few positively stained nuclei in normal taste buds but, in those of mice 1–5 days after denervation, the number of positive nuclei had increased to 3–5 times that of taste buds from normal mice. Electron-microscopic observation after denervation demonstrated taste bud cells containing condensed and fragmentary nuclei in a cytoplasm with increased density. The results show that taste bud cells under normal conditions die by apoptosis at the end of their life span, and that gustatory nerve sectioning causes apoptosis of taste bud cells with taste buds decreasing in number and ultimately disappearing.

Expression of E- and P-cadherin during tooth morphogenesis and cytodifferentiation of ameloblasts

Abstract Cell-cell adhesion is fundamental in morphogenesis and is known to be mediated by several groups of cell adhesion molecules. Cadherins are a group of such molecules involved in the Ca2+-dependent cell-cell adhesion mechanism and are found in most kinds of tissue. In this study using indirect immunofluorescence microscopy, we analyzed the distribution of two kinds of cadherins, E- and P-cadherin, in developing tooth germs. In the molar tooth germs at the early bud stage, marginal cells of the epithelial tooth bud expressed both E- and P-cadherin, whereas central cells expressed only E-cadherin. At the cap stage, in addition to the cells of the inner and outer enamel epithelium, which outline the enamal organ, cells of the enamel knot, which is thought to control tooth morphogenesis, strongly expressed P-cadherin. The expression of P-cadherin was prominent in the inner enamel epithelium during the early to mid bell stage, and was also evident in the non-dividing cell masses at future cusp tips, which are the so-called secondary enamel knots. In the tooth germ at the late bell stage when the cells of the inner enamel epithelium began to polarize to differentiate into ameloblasts, the polarizing ameloblasts lost P-cadherin and strongly expressed E-cadherin. However, E-cadherin was also lost from polarized ameloblasts at later stages. The stratum intermedium and the stellate reticulum were E-cadherin positive from the bell stage onward even at the stages when the ameloblasts became E-cadherin negative again. These results suggest that the differential expression of E- and P-cadherin during morphogenetic stages plays a role in the regulation of tooth morphogenesis, whereas alteration of E-cadherin expression during later stages of tooth development is related to differentiation and function of the ameloblasts and other cells supporting amelogenesis.

Expression of neural cell adhesion molecule (NCAM) during the first molar development in the mouse

NCAM, the neural cell adhesion molecule, was immunolocalized in the mandibular first molar tooth germ of the mouse. NCAM was first detected in the tooth germ of the late bud stage, where only the cells in the outer part of the condensed mesenchyme (primitive dental follicle) exhibited faint immunoreactivity. The entire dental follicle was intensely immunostained for NCAM from cap stage to the stage when root formation started. During root formation, NCAM disappeared from the follicular tissue surrounding the cervical root as well as from the part covering the crown top. This loss of NCAM proceeded in the direction of the root apex, but even after the tooth had achieved functional occlusion, NCAM was still expressed by the mesenchymal cells adjacent to the root apex. On the other hand, NCAM was negative in the dental papilla until birth. After birth, NCAM-immunoreactivity appeared in the basal portion of the dental papilla, but this NCAM-positive area gradually diminished in width during the root elongation. Instead, another NCAM-positive zone appeared in the core of the pulp during root formation. Even in the tooth that had already erupted, the pulp core contained cells that were strongly positive for NCAM immunostaining. In addition to its expression in the above two mesenchymal cell lineages, NCAM was transiently expressed by epithelial components of the tooth germ, some of the cells of the dental lamina and the enamel organ. The results suggest that NCAM participates in several processes of tooth development.

Keratin filaments of epithelial and taste-bud cells in the circumvallate papillae of adult and developing mice.

SummaryKeratin filaments of epithelial- and taste-bud cells in the circumvallate papillae of adult and developing mice were studied by immunocytochemistry using monoclonal antikeratin antibodies (PKK2 and PKK3) and by conventional electron microscopy. Elongated cells (type-I,-II, and-III cells) of the taste buds were stained by PKK3 antibody, which reacts with 45-kdalton keratin, whereas basal cells of the taste buds and surrounding epithelial cells showed negative staining with PKK3. Such PKK3-reactive cells occurred at 0 day after birth, when taste-buds first appeared in the dorsal surface epithelium of the papillae. Thus 45-kdalton keratin seems to be an excellent immunocytochemical marker for identifying taste-bud cells. Epithelial cells in all layers of the trench wall and basal layer cells of the dorsal surface contained densely aggregated bundles of keratin filaments that reacted with PKK2 antibody, but not with PKK3. In contrast, taste-bud cells and spinous and granular layer cells of the dorsal surface possessed loose aggregated bundles of filaments that reacted with PKK3, but not with PKK2. These results suggest that the aggregation and distribution pattern of keratin filaments may reflect differences in the keratin subtypes that comprise these filaments.

Gene expression of β–catenin is up-regulated in inner dental epithelium and enamel knots during molar tooth morphogenesis in the mouse

Beta–catenin is a multi–functional molecule that is involved in both cell–cell adhesion and signaling. We analyzed changes in β–catenin gene expression during mouse molar tooth development by in situ hybridization. Prominent up–regulation of the expression of this gene was evident exclusively in the enamel knot at the early cap stage. During the cap and bell stages, the enamel knot, inner dental epithelium, and differentiating stratum intermedium expressed the β–catenin gene more strongly than other parts of the enamel organ. During these stages, the strength of the gene expression changed heterogeneously within the inner dental epithelium and stratum intermedium. However, the heterogeneity was not evident at the late bell stage, when the cells in the inner dental epithelium had differentiated into ameloblasts at the cusp tip. No spatiotemporal change in β–catenin gene expression was apparent in the dental papilla except for the cells that differentiated into odontoblasts, which became negative for the expression of the gene after their differentiation. Thus, the up-regulated expression of the β–catenin gene was strongly associated with epithelial morphogenesis. These findings raise the possibility that the up–regulation of the gene expression and the stabilization of the protein by Wnt signaling play a role in the regulation of the activities of β–catenin in tooth morphogenesis.

Olfactory epithelium consisting of supporting cells and horizontal basal cells in the posterior nasal cavity of mice

Abstract. The olfactory epithelium of mice generally consists of olfactory cells, progenitors of olfactory cells (globose basal cells), supporting cells, and horizontal basal cells. However, in the dorsal fossa (the roof) of the posterior nasal cavity of mice, we found seven epithelial patches consisting of only non-neuronal cell types, i.e., supporting cells and horizontal basal cells, among the normal olfactory epithelium. The supporting cells occupied three or four layers in the apical to middle regions; in the basal region, horizontal basal cells were localized in a single row adjacent to the basement membrane. Bowmans gland ducts were also present in the epithelium. Neuronal cells (olfactory cells and globose basal cells) were totally absent. The ultrastructure of the supporting cells, horizontal basal cells, and Bowmans glands was essentially similar to that in the normal olfactory epithelium. In the early postnatal period (P1–P7), cell types in the epithelium were the same as those in the normal olfactory epithelium. From P10 to P21, olfactory cells and globose basal cells had disappeared from the olfactory epithelium. At this period, the number of TUNEL-positive cells was significantly higher than that in the surrounding olfactory epithelium; ultrastructurally, many apoptotic figures were observed. This suggests that the epithelium consisting of supporting cells and horizontal basal cells is generated by the apoptotic death of olfactory cells and globose basal cells during postnatal development.

Expression of GDNF and GFRα1 in mouse taste bud cells

GDNF (glial cell line‐derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFRα1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or α‐gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFRα1, because NCAM is reported to be expressed in type‐III cells, PGP 9.5, in type‐III and some type‐II cells, and α‐gustducin, in some type‐II cells. Normal taste bud cells expressed both GDNF and GFRα1. The percentage of GDNF‐immunoreactive cells among all taste bud cells was 31.63%, and that of GFRα1‐immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF‐immunoreactive cells in the taste buds were reactive with anti‐NCAM or anti‐PGP 9.5 antibody, but could be stained with anti‐α‐gustducin antibody. On the other hand, almost all anti‐PGP 9.5‐ or anti‐α‐gustducin‐immunoreactive cells were positive for GFRα1. Thus, GDNF‐immunoreactive cells did not include type‐III cells, but type‐II cells, which are α‐gustducin‐immunoreactive; on the other hand, GFRα1‐immunoreactive cells included type‐II and ‐III cells, and perhaps type‐I cells. We conclude that GDNF in the type‐II cells may exert trophic actions on type‐I, ‐II, and ‐III taste bud cells by binding to their GFRα1 receptors. J. Comp. Neurol. 479:94–102, 2004.

Subcellular localization of β-catenin and cadherin expression in the cap-stage enamel organ of the mouse molar

We analyzed the subcellular distribution of β-catenin in the cap-stage enamel organ and compared it with the expression of E- and P-cadherin by using confocal laser microscopy. The amounts of the molecules in the cytoplasm and the nucleus showed regional variations in the enamel organ, whereas cell surface-associated β-catenin was ubiquitous. In both the enamel knot and the inner dental epithelium, β-catenin was detected in the cytoplasm and in the nucleus. However, the amount of nuclear β-catenin was apparently higher in the enamel knot than in the inner dental epithelium. P-cadherin also gave a stronger signal in the enamel knot than in other parts of the enamel organ. In the stellate reticulum, where E-cadherin was preferentially expressed, as well as in the cervical loop and outer dental epithelium, β-catenin was localized in the cytoplasm but not in the nucleus. The nuclear localization of β-catenin in the enamel knot suggests a specific activation of the canonical Wnt signaling pathway. A coincident upregulation of P-cadherin was observed in this area. Altogether, these observations suggest the possibility of a linkage between cell adhesion and Wnt signaling in the enamel knot.

Expression of the neural cell adhesion molecule (NCAM) during second- and third-molar development in the mouse

Distribution of the neural cell adhesion molecule (NCAM) during the development of the mandibular second- and third-molars of the mouse was studied by indirect immunofluorescence techniques. At the initial stage, NCAM was intensely expressed by the mesenchymal cells surrounding the dental lamina, and by the cap stage NCAM expression by the mesenchymal cells became restricted to the dental follicle. After that, in addition to the follicular mesenchyme, some cells in the basal part of the dental papilla showed NCAM-immunoreactivity for a while after the hard tissue formation had started. During root formation, the follicular cells lost NCAM first from the level of the cervical root and later from the coronal part, while an additional NCAM positive area appeared deep in the dental papilla. Even after the teeth had erupted, NCAM was expressed in the tissue surrounding the apical root and in the pulp core. During the initial and bud stages, the pattern of NCAM expression in the second and third molars was different from that in the first molar, where NCAM was found only after the late bud stage; while from the cap stage onward, it changed in the same sequence as in the first molar. The different pattern of NCAM expression implies that there is a difference in developmental events between the early stages of the first and the other two molars. On the other hand, the common sequence of NCAM expression in the tooth germs later than the cap stage suggests that NCAM plays an essential role in the formation of the basic structure of the teeth and periodontal tissues.

Expression of neural cell-adhesion molecule mRNA during mouse molar tooth development.

This study employed in situ hybridisation using a probe recognising all isoforms of the molecule. Expression of the molecule in tooth germs started at embryonic day 13, when they were at the bud stage. Both inner cells of the epithelial bud and peripheral cells of the dental mesenchyme were positive. At the cap stage, positive cells were found in the inner part of the enamel organ but only in a limited area near the outer enamel epithelium. In the mesenchyme at the cap stage, expression was weak in the dental papilla and strong in the follicle. From the bell stage onward, epithelial cells in the enamel organ were negative except for the cells of the stratum intermedium, which were transiently positive at early and late bell stages. In the dental papilla, expression had mostly ceased during and after the bell stage, although transient expression was found in cuspal areas at the early bell stage. The dental follicle strongly expressed neural cell-adhesion molecule (NCAM) to the end of the experimental period, at post-natal day 4. In contrast to the first molar at its earliest stage of appearance, in which both the thickened epithelium and surrounding mesenchyme were negative for the expression of the molecule, the second molar appeared as a combination of extending epithelial thickenings and mesenchymal cells strongly positive for its expression. This study newly identifies the dental papilla and the stratum intermedium as NCAM-expressing sites.